The present invention relates to methods of using the three-dimensional quadrupole ion trap mass spectrometer ("ion trap") which was initially patented in 1960 by Paul, et al., (U.S. Pat. No. 2,939,952). In recent years use of the ion trap mass spectrometer has grown dramatically, in part due to its relatively low cost, ease of manufacture, and its unique ability to store ions over a large range of masses for relatively long periods of time.
The quadrupole ion trap comprises a ring-shaped electrode and two end cap electrodes. Ideally, both the ring electrode and the end cap electrodes have hyperbolic surfaces that are coaxially aligned and symmetrically spaced. By placing a combination of AC and DC voltages (conventionally designated "V" and "U", respectively) on these electrodes, a quadrupole trapping field is created. A trapping field may be simply created by applying a fixed frequency (conventionally designated "f") AC voltage between the ring electrode and the end caps to create a quadrupole trapping field. The use of an additional DC voltage is optional, and in commercial embodiments of the ion trap no DC voltage is normally used. It is well known that by using an AC voltage of proper frequency and amplitude, a wide range of masses can be simultaneously trapped.
The mathematics of the quadrupole trapping field created by the ion trap are well known and were described in the original Paul, et al., patent. For a trap having a ring electrode of a given equatorial radius r.sub.0, with end cap electrodes displaced from the origin at the center of the trap along the axial line r=0 by a distance z.sub.0, and for given values of U, V and f, whether an ion of mass-to-charge ratio (m/e, also frequently designated m/z) will be trapped depends on the solution to the following two equations: ##EQU1## where .omega. is equal to 2.pi.f.
Solving these equations yields values of a.sub.z and q.sub.z for a given ion species having the selected m/e. If the point (a.sub.z, q.sub.z) maps inside the stability envelop, the ion will be trapped by the quadrupole field. If the point (a.sub.z, q.sub.z) falls outside the stability envelop, the ion will not be trapped and any such ions that are introduced within the ion trap will quickly move out of the trap. By changing the values of U, V or f one can affect the stability of a particular ion species. Note that from Eq. 1, when U=0, (i.e., when no DC voltage is applied to the trap), a.sub.z =0.
(It is common in the field to speak in abbreviated fashion in terms of the "mass" of ions, although it would be more precise to speak of the mass-to-charge ratio of ions, since that is what really affects the behavior of an ion in a trapping field. For convenience, this specification adopts the common practice, and generally uses the term "mass" as shorthand to mean mass-to-charge ratio.)
The typical method of using an ion trap consists of applying voltages to the trap electrodes to establish a trapping field which will retain ions over a wide mass range, introducing a sample into the ion trap, ionizing the sample, and then scanning the contents of the trap so that the ions stored in the trap are ejected and detected in order of increasing mass. Typically, ions are ejected through perforations in one of the end cap electrodes and are detected with an electron multiplier.
A number of methods exist for ionizing sample molecules. Most commonly, sample molecules are introduced into the trap and an electron beam is turned on, ionizing the sample within the trap volume. This is referred to as electron impact ionization or "EI". Alternatively, ions of a reagent compound can be created within or introduced into the ion trap to cause ionization of the sample due to interactions between the reagent ions and sample molecules. This technique is referred to as chemical ionization or "CI". Other methods of ionizing the sample, such as photoionization using a laser beam or other light source, are also known. For purposes of the present invention the specific ionization technique used to create ions is generally not important.
The various known ionization techniques all involve what will be referred to as "ionization parameters" that effect the number of ions created or introduced into the ion trap. In turn, the number of ions stored within the trap volume determines the space charge within the trap, since the space charge in the trap is a function of the overall ion population. Various ionization parameters may be used to control the number of ions introduced in the trap depending on the specific method of ion introduction. For example, when using El, the number of ions created in the trap is a function of the intensity of the electron beam used to create the ions as well as the length of time the beam is turned on. Thus, both of these are ionization parameters as that term is used in the present specification, since the ion population in the trap can be controlled by varying the intensity of the beam or by varying the length of time the beam is turned on. Likewise, when using photoionization, both the length of time the light beam is turned on and the intensity of the beam are considered ionization parameters.
When using CI, the reaction time between the sample molecules and the reagent ions is an ionization parameter. It is noted that reagent ions are normally created within the ion trap by ionizing reagent molecules using an electron beam. In other words, the reagent ions are normally created by EI. In such a situation, the quantity of reagent ions created in the ion trap is dependent on the same ionization parameters described above, i.e., the length of time the electron beam is turned on and the intensity of the beam. When ionizing reagent ions, measures are normally taken to eliminate any sample ions simultaneously formed in the ion trap. According to the present invention, another method of creating reagent ions for a CI experiment is to allow initial precursor ions to react with a reagent gas to form the desired reagent ions. Thus, the reagent ions are themselves formed by chemical ionization.
While in most instances sample ions are created within the trap volume, in some instances ions may be created externally by any of the foregoing methods and transported into the ion trap using known ion transport means. In such instances, an electronic gating arrangement may be used to control the flow of ions into the trap, and the length of time the ion gate is "open" can be used to control the ion population introduced into the ion trap. Thus, this would also be considered an ionization parameter according to the present invention.
As described, there are a number of known methods for creating the ions that are trapped in an ion trap. For purposes of this specification, the terms "introduced" and "introducing," when used in connection with sample ions, are intended to cover all of the various methods. Thus, ions may be introduced into the ion trap either by formation within the trap volume, as by traditional in trap I or CI techniques, or by formation outside of the ion trap and transport into the trap volume.
Once ions are introduced into the trap it is generally the object of the spectroscopist to obtain a mass spectrum of the contents of the ion trap, i.e., to determine the mass number and relative abundance of the trapped ions. While some types of experiments require further manipulations of the ion trap prior to obtaining a mass spectrum, such as isolating a "parent" ion and performing an MS/MS experiment, commercial ion traps are most commonly used to obtain mass spectra.
Obtaining a mass spectrum generally involves scanning the trap so that ions are removed from the ion trap and detected. U.S. Pat. No. 4,540,884 to Stafford, et al., describes a technique for scanning one or more of the basic trapping parameters of the quadrupole trapping field, i.e., U, V or f, to sequentially cause trapped ions to become unstable and leave the trap. Unstable ions tend to leave in the axial direction and can be detected using a number of techniques, for example, as mentioned above, a electron multiplier or Faraday collector connected to standard electronic amplifier circuitry.
In the preferred method taught by the '884 patent, the DC voltage, U, is set at 0. As noted, from Eq. 1 when U=0, then a.sub.z =0 for all mass values. As can be seen from Eq. 2, the value of q.sub.z is directly proportional to V and inversely proportional to the mass of the particle. Likewise, the higher the value of V the higher the value of q.sub.z. In the preferred embodiment the scanning technique of the '884 patent is implemented by ramping the value of V. As V is increased positively, the value of q.sub.z for a particular mass increases to the point where it passes from a region of stability to one of instability. Consequently, the trajectories of ions of increasing mass to charge ratio become unstable sequentially, and are detected when they exit the ion trap. This technique will be referred to as mass instability scanning.
According to another known method of scanning the contents of an ion trap, a supplemental AC voltage is applied across the end caps of the trap to create an oscillating dipole field supplemental to the quadrupole field. (Sometimes the combination of a quadrupole trapping field and a supplemental rf dipole field is referred to as a "combined field.") In this method, the supplemental AC voltage has a different frequency than the primary AC voltage V. The supplemental AC voltage can cause trapped ions of specific mass to resonate at their so-called "secular" frequency in the axial direction. When the secular frequency of an ion equals the frequency of the supplemental voltage, energy is efficiently absorbed by the ion. When enough energy is coupled into the ions of a specific mass in this manner, they are ejected from the trap in the axial direction and where they can be detected as has been described. The technique of using a supplemental dipole field to excite specific ion masses is sometimes called axial modulation. As is well known in the art, axial modulation is also frequently used to eject unwanted ions from the trap, and in connection with MS/MS experiments to cause parent ions in the trap to collide with molecules of a background buffer gas and fragment into daughter ions. This latter technique is commonly referred to as collision induced dissociation (CID). As is also well known, whether an ion will be ejected by axial modulation from the trap, or instead merely fragmented, is largely dependent on the voltage level of the supplemental dipole voltage.
The secular frequency of an ion of a particular mass in an ion trap depends on the magnitude of the fundamental trapping voltage V. Thus, there are two ways of bringing ions of differing masses into resonance with the supplemental AC voltage: scanning the frequency of the supplemental voltage in a fixed trapping field, or varying the magnitude V of the trapping field while holding the frequency of the supplemental voltage constant. Typically, when using axial modulation to scan the contents of an ion trap, the frequency of the supplemental AC voltage is held constant and V is ramped so that ions of successively higher mass are brought into resonance and ejected. The advantage of ramping the value of V is that it is relatively simple to perform and provides better linearity than can be attained by changing the frequency of the supplemental voltage. The method of scanning the trap by using a supplemental voltage will be referred to as resonance ejection scanning.
Resonance ejection scanning of trapped ions provides better sensitivity than can be attained using the mass instability technique taught by the '884 patent and produces narrower, better defined peaks. In other words, this technique produces better overall mass resolution. Resonance ejection scanning also substantially increases the ability to analyze ions over a greater mass range.
In commercial embodiments of the ion trap using resonance ejection as a scanning technique, the frequency of the supplemental AC voltage is set at approximately one half of the frequency of the AC trapping voltage. It can be shown that the relationship of the frequency of the trapping voltage and the supplemental voltage determines the value of q.sub.z (as defined in Eq. 2 above) of ions that are at resonance. Indeed, sometimes the supplemental voltage is characterized in terms of the value of q.sub.z at which it operates.
While the most common method of analyzing the contents of an ion trap involves causing ions to sequentially leave the trap in the axial direction where they can be intercepted by an external detector, other detection methods, including in-trap detection methods are well known and may be used in connection with the present invention. Some of these techniques are described below.
Commercially, most ion traps are sold in connection with gas chromatographs (GC's). As is well known, a GC serves to separate a complex sample into its constituent compounds thereby facilitating the interpretation of mass spectra. Of course, ion trap technology is not limited to use with GC's, and other sample input sources are known. For example, with an appropriate interface, a liquid chromatograph (LC) can be used as a sample source. 0f course, for some applications no sample separation is required, and sample may be introduced directly into the ion trap.
The flow from a GC is continuous, and a modern high resolution GC produces narrow peaks, sometimes lasting only a matter of seconds. In order to obtain a mass spectra of narrow peaks, it is necessary to perform at least one complete scan of the ion trap per second. The need to perform rapid scanning of the trap adds constraints which may also affect mass resolution and reproducibility. Similar constraints exist when using the ion trap with an LC or other continuously flowing, variable sample stream.
As with most any instrument of its type, it is known that the dynamic range of an ion trap is limited, and that the most accurate and useful results are attained when the trap is filled with the optimal number of ions. Ion trap mass spectrometers are extremely susceptible to deleterious effects of space charge and ion molecule reactions. The space charge in the ion trap alters the overall trapping field interfering with mass resolution and calibration. Moreover, space charge affects the trapping efficiency and ion molecular reactions. If too few ions are present in the trap, sensitivity is low and peaks may be overwhelmed by noise. If too many ions are present in the trap, space charge effects can significantly distort the trapping field, and peak resolution can suffer.
The prior art has addressed this problem by using a so-called automatic gain control (AGC) technique which aims to keep the total charge in the trap at a constant level. In particular, prior art AGC techniques use a fast "prescan" of the trap to estimate the charge present in the trap, and then uses this prescan to control a subsequent analytical scan. While this approach has been acceptable for many applications and experiments, the inventor has determined that it does not provide highly accurate control over the space charge in the ion trap and, thus, limits the ability to obtain very high resolution.
There are several prior art AGC methods that have been used to control the space charge levels in ion traps so as to optimize the performance of the trap for various applications. These prior art methods all have in common a two-step process of conducting each sample analysis: performing a prescan to estimate the concentration of sample ions present in the trap using fixed, predetermined ionization parameters, followed by an analytical scan of the trap performed using optimized the ionization parameters, based on information obtained from the prescan. The goal of these techniques is to always store approximately the same total number of ions in the trap as the sample levels change. As used herein the term prescan refers to a scan of the contents of the trap which is performed for the purpose of optimizing an ionization parameter. In a prescan, no mass spectrum for use by the spectroscopist is created. A prescan is normally performed so rapidly that meaningful mass spectral data would not be discernable due to the very poor mass resolution associated with rapid scanning. The lack of mass data is not important for a prescan since the purpose of a prescan is simply to measure the amount of charge in the ion trap. Likewise, as used herein the term analytical scan refers to a scan intended to collect mass spectral data of the contents of the ion trap.
In the prior art method of Stafford, et al., (U.S. Pat. No. 5,107,109) the sample concentration in the trap is measured in a prescan by applying a short, fixed-duration electron beam to the trap to cause sample ionization, followed by a rapid measurement of the total ion content (TIC) of the trap. This measurement is used to control the number of sample ions in the ion trap during the subsequent analytical scan. There is no teaching to rid the trap of any unwanted ions during either the prescan or the subsequent analytical scan.
In the prior art method of Weber-Grabau, et al., (U.S. Pat. No. 4,771,172) a fixed-duration prescan is again used, in a manner similar to the method of the '109 patent in conjunction with chemical ionization to measure the sample concentration in the trap prior to the analytical scan. This patent also teaches eliminating unwanted sample ions from the trap during the period in which reagent ions are created in the trap. As in the '109 patent, during the prescan both the length of time that the electron beam is turned on to ionize the reagent ions, as well as the length of time the reagent ions are allowed to react with the sample to ionize it, are fixed.
The prior art method of Kelley (U.S. Pat. No. 5,200,613) also discloses a prescan which uses a short, fixed ionization time as in the method of the '109 patent, with the improvement being the additional step of applying notched-filtered noise to the trap to resonantly eject undesired ions. The ion ejection, by means of filtered noise, to isolate parent ions, is performed in connection with both the prescan and the analytical scan. Kelley also teaches use of this process with MS/MS experiments.
All of these prior art methods suffer from utilizing fixed, predetermined ionization parameters during the prescan step to estimate the sample concentration in the trap and to adjust an ionization parameter during the subsequent analytical scan. However, a variety of ion-molecule reactions can occur within the ion trap which alter the relative ion intensity of sample molecules as described below.
The method of the '109 patent, has the additional limitation in that the prescan measures the integrated ion signal from a broad mass range of ions that are trapped during the ionization period of the prescan. In a complex matrix eluting from a GC the ratio of sample to matrix can change dramatically during the elution of a sample peak from the chromatograph. As will be understood by those skilled in the art, the term "matrix" refers to the entire mixture of compounds that is introduced into the ion trap at any given time and includes molecules different from the sample compound(s) of interest. Such background molecules may be present for a variety of reasons. Thus, fixed ionization conditions during the prescan may increase the error in the sample level determination by including undesired ions from the matrix. Ionization of the matrix will often produce large numbers of ions with masses below that of the parent ion. Low mass ions in particular are troublesome in an ion trap, because they decrease the trapping efficiency of the higher mass parent ions. When very high concentration levels of the matrix are present, use of a fixed prescan may cause the number of sample ions that are trapped to change with the level of the matrix, even if the sample level is constant.
The method of Kelley attempts to reduce the sample/matrix problem by improving upon the method of the '109 patent, by adding the additional step of applying notched filtered noise to the trap during ionization to eject unwanted ions and to isolate a parent ion. Because of the continuous frequency distribution of noise, large power levels are required in order to have enough power at the secular frequency of all unwanted ions in order to eject them completely.
Another technique for controlling the effects of space charge in an ion trap is described in the prior art method of Fies, et al., (U.S. Pat. No. 4,650,999). As described in that patent, control of the space charge in the trap over the trapping range is controlled by using a segmented scan of the ion trap, wherein each segment traps ions within a portion of the mass range by using a different three dimensional quadrupole field. After ions are created and trapped the three dimensional trapping field for each segment is then changed, as taught by the '884 patent, so that trapped ions of consecutive masses become unstable and leave the trap for detection. As stated in the specification of the '999 patent, "Each segment will have different storage voltages and starting mass" (column 4, line 46). In simple terms, the patent teaches periodically interrupting a continuous scan of the trap to form ions for the various scan segments.
There are several significant disadvantages of the prior art method of Fies, et al. For example, it is well known that when using EI, the ionization conditions of the trap vary as a function of the trapping conditions. In effect, the trapping voltages affect the energy of the electron beam used for ionization. The method of Fies, et al., requires that each segment be ionized under different trapping conditions and, hence, the energy of the ionizing electron beam varies from one segment to the next. In addition, there can be undesired ion-molecule reactions that occur even during the time span of the ionization period.
As an example, in a class of compounds known as fatty acid methyl esters (FAME) there is an abundant ion at mass 74 which is a strong proton donor. The presence of FAME ions results in the protonation of the neutral FAME sample molecules (molecular mass =M), with the subsequent formation of the protonated molecular ion of mass equal to M+1. The resulting abnormally high value for the M+1 ion intensity is an undesired result of the ion-molecule reactions that take place during the ionization period and subsequent times prior to the mass 74 ion being scanned out from the trap. The prior art method of Fies makes no provision for eliminating the unwanted ion-molecule reactions that occur during the ionization period.
The method of Fies also does not provide a means of eliminating unwanted masses above the mass range of the particular mass segment that is being scanned; for a particular mass segment, only the space charge from those low mass ions that were scanned out of the trap during the previous segment is removed. A further limitation is the limited range over which the trapping voltage can be adjusted during the ionization period without affecting the trapping efficiency.
The prior art method of Kelley eliminates unwanted ions above and below a selected mass range by using a notched filtered noise signal to resonate the unwanted ions out of the trap. In addition, Kelley teaches use of a prescan to optimize the ionization parameters for an analytical scan. Since the prescan "integrates" the ions in the prescan range it can only be used to optimize the following analytical scan in an "average, integrated" manner. An additional limitation of the use of fixed prescans is the additional time required to perform the ionization and ejection/detection step during the prescan.
A final, and significant limitation of the prior art methods of sampling and controlling the space charge in the trap, relates to the optimization of the detection of low intensity ions in the presence of other, larger intensity ions in the same spectrum. The fixed ionization prescan method would often be unable to detect and thus optimize low intensity ions, as the integrated prescan ion intensity would be mostly due to the intense ion and thus the optimization of the following analytical scan would be done mostly for the high intensity ion. To the extent that the technique of Fies, et al., has been combined in the prior art with the prescanning technique of the '109 patent, prescanning has not been conducted separately as to each mass segment. Rather prescanning has been used only to determine the TIC of the total mass range in the ion trap.